| Onchidium reevesii is an intertidal mollusk widely distributed on the southeastern coast of China.It evolved a special ability to avoid tide water due to its limited ability to withstand hypoxia underwater,.The tide emits low-frequency sounds of 5-200 Hz.The special ability to avoid tides is due to low-frequency hearing or long-term memory is not yet conclusive.At the same time,the ganglion of O.reevesii is mature and easy to obtain,which is a good material for researching memory and low-frequency hearing-related genes.So far,there is no screening study on low-frequency hearing related genes of O.reevesii.In this paper,the genes related to auditory brainstem response were selected from the mouse phenotype database.We compare those date with transcript of O.reevesii treated with 100 Hz,300Hz,and500 Hz low-frequency sound.The preliminary screening of low-frequency sound was carried out to obtain OrNXN,a gene that may be related to low-frequency hearing in the O.reevesii.The c DNA sequence of OrNXN gene was cloned.Then we verify whether it is differentially expressed under low-frequency sound processing by QPCR technology.,and combined with the tide to explore its function of perceiving low-frequency sound to avoid the tide.At the same time,the tissue distribution and expression level under tidal stress of the memory-related gene OrPKA were studied.This experiment conducted a preliminary exploration of the unique low-frequency sound perception ability produced in the evolution and development of the shellfish.We pave the way for the in-depth study of the evolution of low-frequency hearing-related genes,at the same time,we have tried to understand the mechanism of the tidal memory ability of O.reevesii.1.Cloning and expression analysis of OrNXN gene associated with low frequency hearing in O.reevesiiThe genes with significantly different expressions were selected from the different frequency sound gene transcriptomes of the O.reevesii.We compared these genes with the genes related to human auditory perception and deafness in IMPC,and the hearing-related gene NXN was discovered.The c DNA sequence of OrNXN gene was cloned by rapid amplification of c DNA ends(RACE)technology,and the sequence was analyzed by bioinformatics;real-time quantitative PCR(q PCR)technology was used to analyze the relative expression in different tissues,different frequency sounds,and different tide nodes.The results show that the length of OrNXN is 1593 bp with 432 bp of open reading frame.OrNXN protein is encoded by 143 amino acids.The molecular weight of OrNXN is 15.57 kd.Domain prediction analysis shows that OrNXN contains a typical sulfur redox protein structure of the thioredoxin family.The results of multiple sequence alignment and the phylogenetic tree construction showed that OrNXN has high similarity with other species.The q PCR results show that OrNXN is expressed in the intestine,ganglia,gonads and gastropods of O.reevesii.The expression is highest in the intestine,followed by ganglia.The results indicate that OrNXN may play a special function in the intestinal tract,and may be involved in the development of early neural organs in the ganglia.Patients with low-frequency hearing impairment often have abnormal ABR peaks in the auditory organs.OrNXN may be involved in the development of the auditory organs,indirectly associated with low frequency sounds.2.Expression of OrNXN gene under low frequency hearing and tidal stimulation in O.reevesiiThe low-frequency sound of the tide is roughly within 200 Hz.The high tide point and the low tide point generally have a time interval of about 6 hours.Four low-frequency sounds of 50,100,150,and 200 Hz are used to stimulate O.reevesii continuously for 6 hours.On July 25,which is spring tide of July,we sample O.reevesii.We sample seven timing points which are highest tide point,the lowest tide point,the rising tide point and the ebb tide point.These 7 points represent a complete tide.The results showed that OrNXN was differentially expressed at 200 Hz and 50 H under different sound frequency stimulation(P<0.05).The relative expression level of OrNXN is different at different tidal nodes,it basically changes with the tide water level.Studies have shown that OrNXN may respond to the low-frequency sounds emitted by the tide when O.reevesii is involved in sensing the tidal rhythm.The expression level of high point at the nighttime is lower than that at the daytime.Maybe the distribution of OrNXN in the photoreceptor of the O.reevesii.resulting in differences expression under different illumination.3.Study on how memory-related gene OrPKA response to tidal rhythmWe obtain OrPKA sequence from transcript and verified it.QPCR primers were designed to detect the relative expression of the memory-related gene OrPKA in the tissue of O.reevesii and tidal environment.After 1 month and 2 months of temporary culture in the laboratory,we detected the expression of OrPKA in tidal environment to exam whether memory ability of O.reevesii changes.The results of different expression in tissues showed that OrPKA gene has highest expression in the bisexual glands,was also highly expressed in the ganglia and stomach.The results of tidal stress showed that the expression of OrPKA was consistent with the trend of the tide.First time,the highest expression was at the high tide point and the lowest expression was at the low tide point.After 1 month,the expression level of OrPKA different from the tide trend,the expression level at the low tide point was the highest and the expression level at the high tide point was the lowest.After 2 months the expression of OrPKA was only the highest at low tide,which was quite different from the first experiment.It is speculated that the memory-related gene OrPKA may play a role in reproduction rather than memory after 1 month.Maybe the increased expression of OrPKA during the ebb tide of the second month is owing to the involvement of OrPKA in the oviposition of the gonads.The reason for increase expression of OrPKA during high tide is that maybe after being placed in the tidal environment for 6 hours,the memory-related functions of O.reevesii began to work again.We screened the low-frequency hearing-related gene OrNXN in O.reevesii,cloned its full length,quantified its tissue,and further verified the low-frequency hearing function of OrNXN.The expression of OrNXN under tidal stress is consistent with the tide.One of the reason O.reevesii senses the tide may be hearing the low-frequency sound of the tide.However,O.reevesii may forget the tide after 1month and 2 months of temporary culture in the laboratory.Under normal circumstances,O.reevesii’s perception of the tide should be formed by the combination of hearing and memory. |
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